Repetitive apnea during wakefulness is a cardinal and potentially life-threatening symptom of Rett syndrome (RTT). A recent clinical trial reveals that many of these apneic events in RTT patients result from behaviorally- induced involuntary breathholding (cessation of expiration) instead of chemoreflex-mediated central apnea (cessation of inspiration), and that such breathing disturbances are ameliorated by IGF-1 drug treatment to restore BDNF signaling in these patients. Emerging evidence from mutant mouse models of RTT suggests that such pathologic breathholding likely represents a form of recurrent laryngospasm (paroxysmal laryngeal adductor hyperactivity) caused by sensitization of neurons in the Klliker-Fuse nucleus (KFN), a pontine nucleus which plays an important role in controlling laryngeal adductor activity during the post-inspiratory (post- I) phase of the respiratory rhythm and during behavioral tasks such as vocalization and breathholding. A hallmark of the post-I driver neurons in KFN is their critical dependence on NMDA receptor activity, a property which distinguishes these neurons from many other central neurons that are excited mainly by AMPA receptor- mediated currents. A central hypothesis to be tested in this proposal is that KFN post-I driver neurons are the site where ascending input from superior laryngeal nerve (SLN) mediating the laryngeal adductor reflex and descending input via midbrain periaqueductal gray (PAG) mediating behavioral control of glottic closure are integrated at the systems level, and that hyperactivity of these KFN neurons due to impairment of a form of NMDA and BDNF receptors-dependent synaptic plasticity may underlie the behaviorally-evoked laryngospasm in RTT at the cellular level. As a baseline (Aim 0), we will examine whether IGF-1 treatment ameliorates the breathholding phenotype during wakefulness in a mutant mouse model of RTT. Aim 1 will investigate the role of KFN post-I driver neurons in modulating the laryngeal adductor reflex induced by ascending SLN input in mutant and wild-type mice with or without IGF-1 treatment. In Aim 2, we will investigate the role of these KFN neurons in modulating the laryngeal adductor response induced by descending PAG input in these animals. These multisensory integration data will be subjected to a novel multiscale fingerprinting assay which verifies functional connectivity of the neurons that are upstream and downstream of the PAG-KFN-laryngeal post-I motoneuron pathway by matching a set of timing-, response- and cellular-specific markers shared by these neurons. In Aim 3, we will employ a multi-labeling technique with juxtacellular labeling/single unit recording combined with anterograde axonal tracing to map convergent inputs from PAG and KFN to laryngeal adductor motoneurons in medulla. By comparing these structure-function data from mutant mice and wild-type mice, our goal is to map the central circuits involved in coordination of reflex and volitional control of glottic closure in health and in RTT. Results of this project will inform ongoing and future clinical trials to evaluate the validity of breathholding vs. central apnea as possible outcome measures for assessing drug treatments of RTT patients.